Ischemia-reperfusion injury (IRI) remains the primary obstacle limiting the success of orthotopic liver transplantation (OLT) in patients with end-stage liver disease and those with tumors of hepatic origin. Our group has pioneered the concept that hepatic IRI, an exogenous Ag-independent, innate immune-dominated sterile inflammation response, requires activated CD4+ T cells to facilitate tissue damage. T cell Immunoglobulin Mucin (TIM)-3 receptor has been recognized as a central regulator of T cell activation in a number of auto- / allo-immunity pathologies and organ transplantation. We reported that disruption of TIM-3 - Gal-9 pathway exacerbated hepatocellular injury in mouse livers subjected to warm IR. Then, we found that recipient CD4+TIM-3+ cells conferred resistance against liver IRI, suggesting a discrete host T cell subset should be spared while applying T cell-targeted therapy in transplant recipients. This proposal explores the function of TIM-3 signaling pathway in the mechanism of hepatic IRI in a clinically relevant mouse model of extended cold storage followed by orthotopic liver transplantation (OLT). First, we found that stressed hepatocytes express Gal-9 and HMGB1, i.e., known TIM-3 ligands. Second, we discovered robust expression of TIM-3 on activated liver endothelial cells (LEC). These preliminary data have led us to a central hypothesis that negative regulation between hepatocellular-derived Gal-9/HMGB1 and TIM-3 expressed on host circulating CD4+ T cells/graft LEC is essential to control tissue injury, and impose cytoprotective phenotype in IR-stressed OLT. Two interlocked specific aims will test this hypothesis: Aim 1: Define molecular mechanisms by which hepatocyte Gal-9 - CD4+ T cell TIM-3 negative regulation confers OLT resistance against IR stress. Aim 1.1. Hypothesis: Gal-9 - TIM-3 signaling triggers anti-oxidant response in which amplified Nrf2 activity represses macrophage NF?B/inflammation responses. Aim 1.2. Hypothesis: Gal-9 - TIM-3 signaling enhances hepatocyte autophagy via Keap1/Nrf2 redox network. Aim 2: Define molecular mechanisms by which hepatocellular HMGB1 - endothelial TIM-3 negative regulation alleviates IRI in OLT. Aim 2.1. Hypothesis: HMGB1 conditioning prior to IR insult triggers activation of liver endothelial TIM-3 to repress macrophage trafficking and sequestration in OLT. Aim 2.2. Hypothesis: Hepatocellular HMGB1 - endothelial TIM-3 signaling promotes LEC protective phenotype. These studies will discern novel mechanisms at the innate-adaptive immune interface, which control organ damage/promote homeostasis in IR-stressed OLT; and should contribute to the development of new therapies to increase donor organ pool and improve clinical outcomes.